[Histonet] HT exam

Thu Sep 28 05:13:19 CDT 2006

Oh, boy. One of my least favorite things to try to teach. I'm sure I'll
raise a few hackles with my view on this. (Going to be long, so skip if not
interested.)

Coagulative and non-coagulative fixation concept goes back decades and
decades, maybe even a century or so. My feeling is, it was more an
observation of what the tissue/proteins "physically" seemed to be doing when
placed in a chemical, rather than the actual chemical bonding that we now
talk about.

I demonstrate it to my students using raw egg whites. I get a bunch of
lidded jars, and put about 20 mL of 10% NBF, 20% formalin, zinc-formalin,
alcoholic formalin, 100% alcohol, acetone, Bouin, DB (alcoholic Bouin),
acetic acid. Then I add 1 mL of egg white into each container, and we watch
what happens and how quickly.

- In the 10% and 20% formalin, the egg white remains "loose" - even days
later, I can still swirl it with a small wooden pick. 
- The egg hardens up immediately in the 100% alcohol, acetone, Bouin, DB. 
- The zinc-formalin, alcoholic formalin and the acetic acid harden up slower
- in a few hours, and end up harder than the 10-20% formalin, but softer
than the other chemicals.

Therefore, based on a bunch of experiments like this, it was decided that
the following reagents are non-coagulative for proteins:
- formalin/formaldehyde in any percent
- all aldehyde fixatives (gluteraldehyde, paraformaldehyde)
- potassium dichromate
- osmium tetroxide (doesn't bind to protein, only lipids, so it does not
coagulate proteins)

All other chemicals were considered coagulative for proteins (acetic acid is
a little of both, coagulative for nuclei, non-coagulative for cytoplasm, so
it depends which author you are reading, as to whether acids are coagulative
or non-coagulative. I lump it with coagulative.).

Now, if you have a mixture, say alcoholic-formalin, since the alcohol is
considered coagulative, while the formalin is non-coagulative, then the
mixture is still considered coagulative. If there is even just one chemical
that is coagulative in a fixative mixture and all the rest are
non-coagulative, then that mixture is still considered coagulative. The egg
white does coagulate in alcholic-formalin, but just at a slower rate.

So, for the HT/HTL exam, learn that aqeous solutions that contain only
formalin, potassium dichromate and/or osmium are the only non-coagulative.
If the fixative contains any other reagent, it is now considered
coagulative. (Example - Orth contains only potassium dichromate,
formaladehyde and water. Since both are non-coagulative, Orth is
non-coagulative. Helly contains potassium dichromate, formaldehyde, mercuric
chloride and water. Even though the potassium dichromate and formaldehyde
are non-coagulative, because of mercuric chloride is coagulative, the entire
Helly fixative is considered coagulative (and back when I used to do the egg
white test using mercuric chloride fixatives, yes, the egg white did
coagulate).)

I still teach this concept to my students, as it is in the textbooks and on
the HT/HTL exams. I tell them to memorize it for my fixative test and then
study it again for the ASCP exam, and then they can forget it after that. I
personally don't think it is very helpful information when we are dealing
with amino acids being cross-linked by fixatives. After all, even though the
egg white in the formalin never hardens, I know the egg white proteins are
fixed. And I am more concerned about how the dyes will react now that I've
cross-linked different proteins, rather than "how much did the tissue become
hard".

I, and I think most other program directors of HT/HTL programs, prefer to
teach the additive/non-additive concept, which is also the the ASCP exams. 

Additive means the chemical becomes physically bound (linked) to something
in the tissue. Formalin, which is negatively charged, become cross-linked to
positively charged amino acids (of which there are a lot in cytoplasm and
fewer in nuclei). The overall effect is that with more positively charged
amino acids being bound to the formaldehyde molecule, the tissue is now
slightly more negatively charged. On the other hand, zinc and mercuric
salts, which are positively charged, will bind to negatively charged amino
acids (of which there are fewer in cytoplasm and more in nuclei). So because
the metal salts are binding to positive amino acids, the tissue has more
negative amino acids still available(nothing bound to them), so the tissue
is now slightly more positively charged. All of this helps to explain why
tissues stain differently with H&E (charged dyes pick up differently), IHC
(different cross-links at different amino acids) and look different (nuclear
preservation, for example).

If this is a little hard to see in your mind, imagine 6 positive charge and
6 negative charges. Net charge is zero. Now bind a negative aldehyde to two
of the positive charges. What we have left is 4 positives and 6 negatives.
Net charge is more negative. That's how the aldehydes work. Zinc and mercury
are the opposite. Being positively charged, they will bind 2 negative
charges, yielding 6 positive and 4 negatives, net = more positively charged.

There are basically two families of non-additives:

One are the "dehydrants" - any alcohol (ethanol, methanol, etc.), acetone,
etc. They remove water from the tissue, from between the proteins. This
causes the proteins to shrink together. Now a positive and a negative amino
acid (that used to be separated from each other by the water in the tissue)
are moved closer to each other. Now, these two amino acids can bind to each
other. The "dehydrants" are NOT chemically bound to the tissue. Chemically,
since a positive and a negative bind to each other, there is no real change
in the overall charge of the tissue/protein. (Start with the 6 + and 6 -,
bind one + and one -, end result is 5 + and 5 -, so net charge is still
zero.) So those lab that have shortened formalin fixation and their tissues
are mostly alchohol fixed (no change in charges), their stains are going to
look different than if their tissue was completely formalin fixed (tissue
more negatively charged if formalin fixed, no change in charge if primary
fixative was alcohol).

Acids are the other non-additive. They are sort of the opposite of the
dehydrants. They break open the naturally occuring protein bonds that occur
when one positive amino acid is close to a negative amino acid. When these
bonds break, these charged amino acids are now free to bind to water
molecules. The proteins now swell. But the acids are NOT chemically bound to
the tissue or protein.

However, if there is a mixture, we do NOT lump all the chemicals together
and CANNOT say the mixture is either additive or non-additive. We say what
each separate chemical is doing. Zenker then would be: mercuric chloride =
additive; potassium dichromate = additive; acetic acid = non-additive. 

In both additive/non-additive and the coagulative/non-coagulative, the water
and any buffering salts are NOT considered.

So the easy way to remember all this:

Non-coagulative = APDO = any aldehyde, potassium dichromate, osmium
Coagulative = all the rest
Mixture = if there is even one coagulative, then the mixture is coagulative.

Non-additive = 3 A's = alcohols, acetone, acids
Additive = all other chemicals
Mixture = cannot be lumped into any category. Consider each chemical
separately.

Sorry for being long-winded. These are some hard concepts, and I've actually
simplified it a lot.

Peggy A. Wenk, HTL(ASCP)SLS
William Beaumont Hospital
Royal Oak, MI 48073

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Patsy Ruegg
Sent: Wednesday, September 27, 2006 3:30 PM
To: 'Histonet'
Cc: queensd54 <@t> yahoo.com
Subject: [Histonet] HT exam

Can anyone field this question?  Please reply directly to Pat Barnes at
queensd54 <@t> yahoo.com 

My student and I have been searching for information for some time on the
explanation of coagulant -non coaglant fixatives. Sheehan and Carson have
opposite definitions and all of our other material is ambiguous. Can you
help me shed some light on what could be the best answer if it is on his
test Friday? All opinions are welcome. Thanks. Pat

Life is short. Enjoy daily. Shalom.

Patsy Ruegg

IHCtech

12635 Montview Blvd. Ste.215

Aurora, Colorado 80045

Phone: 720-859-4060

Fax: 720-859-4110

pruegg <@t> ihctech.net

www.ihctech.net 

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